DISSECTING THE FUNCTIONAL AND BIOLOGICAL SIGNIFICANCE OF E3 UBIQUITIN LIGASE TRIP12

Abstract

This thesis focuses on identifying the functional and biological significance of an E3 ubiquitin ligase called TRIP12, where the main aim of first half of the thesis is to understand the mechanism of mitotic cell death regulation in colorectal cancer cell lines by TRIP12. FBW7 mutations confer Taxol resistance to cancer cells due to accumulation of its anti-apoptotic substrate MCL-1. We previously identified TRIP12 as a negative regulator of FBW7 protein where we found genetic inhibition of TRIP12 stabilized FBW7 and enhanced FBW7-mediated degradation of its substrate MCL1 in response to Taxol. The precise molecular details of enhanced cell death in response to Taxol in TRIP12 deficient cells remain unexplored this study reveals that Taxol-induced mitotic block in cancer cells is partly controlled by TRIP12 via its positive regulation of MCL-1 protein. It’s also evident that mutating K404/412 lysines of FBW7 to arginine makes it resistant to proteasomal degradation, leading to sharp reduction of MCL-1 protein and sensitizes cancer cells to Taxol-induced cell death in colorectal and ovarian cancer. Apart from its role in overcoming chemoresistance, TRIP12 controls diverse cellular functions. However, the physiological role of TRIP12 in adult tissues is not clear. To address this issue, I used proteomics-based protein-protein interaction studies to identify novel interaction partners of TRIP12 with a view to understand the molecular pathways governed by TRIP12. We identified SMAD4––major regulator of transforming growth factor beta (TGF-β) signaling–– as one of the top interacting partners of TRIP12 and these interactions were further validated and confirmed by IP western blots and GST-pulldown assays. Results of my study confirm that TRIP12 deletion in mammalian cells enhanced TGF-β reporter activity while concomitant deletion of SMAD4 blocked this increase. Finally, I show that TRIP12 regulates TGF-β signaling in Drosophila gut, adult mouse fibroblasts & intestinal organoids, and human cancer cell lines.

Date of Award	2023
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences